A Naturally Occurring Polymorphism in the HIV-1 Tat Basic Domain Inhibits Uptake by Bystander Cells and Leads to Reduced Neuroinflammation

Abstract

HIV-1 Tat protein contributes to HIV-neuropathogenesis in several ways including its ability to be taken up by uninfected bystander CNS cells and to activate inflammatory host genes causing synaptic injury. Here, we report that in the globally dominant HIV-1 clade C, Tat displays a naturally occurring polymorphism, R57S, in its basic domain, which mediates cellular uptake. We examined the effect of this polymorphism on Tat uptake and its consequences for cellular gene transactivation. In decapeptides corresponding to the basic domain, a R57S substitution caused up to a 70% reduction in uptake. We also used a transcellular Tat transactivation assay, where we expressed Tat proteins of HIV-1 clade B (Tat-B) or C (Tat-C) or their position 57 variants in HeLa cells. We quantified the secreted Tat proteins and measured their uptake by TZM-bl cells, which provide readout via an HIV-1 Tat-responsive luciferase gene. Transactivation by Tat-B was significantly reduced by R57S substitution, while that of Tat-C was enhanced by the reciprocal S57R substitution. Finally, we exposed microglia to Tat variants and found that R57 is required for maximal neuroinflammation. The R57S substitution dampened this response. Thus, genetic variations can modulate the ability of HIV-1 Tat to systemically disseminate neuroinflammation.

HIV-1 Tat: Role in Bystander Toxicity

“…Vesicles that originate from this canonical pathway depend on acidification for the maturation of the endosomes which can be perturbed by either keeping cells at 4 degrees Celsius or adding ammonium chloride to prevent acidification. Several studies have shown that Tat uptake is significantly inhibited when cells are subjected to either condition, further demonstrating that Tat uptake is dependent on this pathway (Richard et al, 2005;Ruiz et al, 2019). The second pathway is the caveolar pathway, which is an alternative endocytosis pathway for Tat uptake as it was demonstrated for the uptake of Tat-GFP in HeLa as well as CHO cells (Ferrari et al, 2003;Fittipaldi et al, 2003).…”

“…response RNA element (TAR) of the newly transcribed HIV genomic RNA (Chiozzini and Toschi, 2016). The arginine-rich or basic domain is the second domain of Tat (49-58 aa) that is important for localizing Tat to the nucleus, binding of Tat to TAR element, and the internalization of Tat protein into bystander cells by its interaction with surface proteins such as heparan sulfate proteoglycans (Tyagi et al, 2001;Ruiz et al, 2019). The next domain is the glutamine-rich domain, which has been linked to Tat interaction with the TAR element and important for the Tat-apoptosis function (King et al, 2006;Loret, 2015).…”

“…Tat is secreted by an alternate mechanism that is dependent on its ability to bind to phosphatidylinositol 4,5-bisphosphate (PI (4,5) P2) through a tryptophan residue (W11) and negatively charged patches on the inner leaflet of the plasma membrane (Chang et al, 1997). In addition to secretion, Tat is biologically active both extracellularly and intracellularly when taken up, as seen in multiple studies that performed trans-well transactivation of reporter genes under the control of HIV-1 LTR (Fittipaldi et al, 2003;Vendeville et al, 2004;Fittipaldi and Giacca, 2005;Ruiz et al, 2019).…”

“…It has been shown that Tat with the critical CC motif; mimics chemokines, binds to chemokine receptors, and can recruit monocytes and macrophages to the CNS while Tat without the intact CC motif does not bind to chemokine receptors and it is unable to recruit these cells (Ranga et al, 2004;Mishra et al, 2008). Another molecular signature that has been identified and linked to Tat neurotoxicity is the R57 residue in the basic domain of Tat (Eguchi et al, 2001;Hashida et al, 2004;Ruiz et al, 2019). The basic domain, which is in the same as the cell-penetrating peptide (CPP), mediates cellular uptake of Tat, and the loss of one basic amino acid has been shown by several groups to perturb cellular uptake.…”

“…A recent study has shown, there is a natural polymorphism seen in Tat mostly in HIV-1 subtype C which significantly reduces cellular uptake of Tat with S57. It was equally shown that less cellular uptake of Tat-S57, translated to less induction of pro-inflammation and less neurotoxicity (Ruiz et al, 2019). On the other side, some studies suggest that Tat protein from subtype C is a better transactivator when compared to subtype B (Kurosu et al, 2002;Johri et al, 2015).…”